Genetic circuit design automation for the gut resident species Bacteroides thetaiotaomicron

Taketani, Mao; Zhang, Jianbo; Zhang, Shuyi; Triassi, Alexander J.; Huang, Yu-Ja; Griffith, Linda G.; Voigt, Christopher A.

doi:10.1038/s41587-020-0468-5

Cited by 96 publications

(108 citation statements)

References 64 publications

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“…Epithelial cells obtained from a healthy region of a colon biopsy were expanded as intestinal organoids and seeded as monolayers in Transwells prior to each experiment following the protocol described earlier. 41 Briefly, primary epithelial cells seeded in a Transwell at 268,000 cells per cm 2 (300,000 per well) were allowed to reach confluency in seeding medium for three days, then the cells were switched to differentiation medium for four days before being transferred to GuMI for experiments (Figure 2a). Inside the GuMI platform, the apical compartment was perfused with anoxic apical medium, where the oxygen level was constantly measured at one-minute intervals using probes positioned at the inlet and outlet of the apical compartment during operation with mucosal barrier-microbe cultures.…”

Section: Prediction and Validation Of Oxygen Tensionsmentioning

confidence: 99%

“…The establishment and maintenance of the organoids were done according to the protocols previously described. 41,57 In brief, organoids grown in Matrigel (growth factor reduced, phenol red free; Corning, 356231) droplets were passaged every seven days at a 1:3 split ratio. A medium change was performed on day four after passaging.…”

Section: Colon Organoid Culture and Monolayer Establishmentmentioning

confidence: 99%

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Primary human colonic mucosal barrier crosstalk with super oxygen-sensitiveFaecalibacterium prausnitziiin continuous culture

Zhang

Huang

Yoon

et al. 2020

Preprint

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AbstractThe gut microbiome plays an important role in human health and disease. Gnotobiotic animal and in vitro cell-based models provide some informative insights into mechanistic crosstalk. However, there is no existing system for a chronic co-culture of a human colonic mucosal barrier with super oxygen-sensitive commensal microbes, hindering the study of human-microbe interactions in a controlled manner. Here, we investigated the effects of an abundant super oxygen-sensitive commensal anaerobe, Faecalibacterium prausnitzii, on a primary human mucosal barrier using a Gut-MIcrobiome (GuMI) physiome platform that we designed and fabricated. Chronic continuous co-culture of F. prausnitzii for two days with colon epithelia, enabled by continuous flow of completely anoxic apical media and aerobic basal media, resulted in a strictly anaerobic apical environment fostering growth of and butyrate production by F. prausnitzii, while maintaining a stable colon epithelial barrier. We identified elevated differentiation and hypoxia-responsive genes and pathways in the platform compared with conventional aerobic static culture of the colon epithelia, attributable to a combination of anaerobic environment and continuous medium replenishment. Furthermore, we demonstrated anti-inflammatory effects of F. prausnitzii through HDAC and the TLR-NFKB axis. Finally, we identified that butyrate largely contributes to the anti-inflammatory effects by downregulating TLR3 and TLR4. Our results are consistent with some clinical observations regarding F. prausnitzii, thus motivating further studies employing this platform with more complex engineered colon tissues for understanding the interaction between the human colonic mucosal barrier and microbiota, pathogens, or engineered bacteria.

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Section: Prediction and Validation Of Oxygen Tensionsmentioning

confidence: 99%

Section: Colon Organoid Culture and Monolayer Establishmentmentioning

confidence: 99%

Primary human colonic mucosal barrier crosstalk with super oxygen-sensitiveFaecalibacterium prausnitziiin continuous culture

Zhang

Huang

Yoon

et al. 2020

Preprint

Self Cite

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“…Yet, given the size of our patient cohort and its diversity, further clinical studies are needed to move our method towards the clinic. Interestingly, the bile salt sensing system could also be applied to control the activity of engineered probiotics upon arrival in the gut, as recently proposed in Bacteroides thetaiotomicron 67 .…”

Section: Discussionmentioning

confidence: 99%

A synthetic receptor platform enables rapid and portable monitoring of liver dysfunction via engineered bacteria

Chang

Zúñiga

Conejero

et al. 2021

Preprint

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Bacterial biosensors, or bactosensors, are promising field-deployable agents for medical and environmental diagnostics. However, the lack of scalable frameworks to systematically program ligand detection limits their applications. Here we present a synthetic receptor platform, termed EMeRALD (Engineered Modularized Receptors Activated via Ligand-induced Dimerization) which supports the modular assembly of sensing modules onto a high-performance, generic signaling scaffold controlling gene expression in E. coli. We applied EMeRALD to detect bile salts, a biomarker of liver dysfunction, by repurposing sensing modules from enteropathogenic Vibrio species. We improved the sensitivity and lowered the limit-of-detection of the sensing module by directed evolution. We then engineered a colorimetric bactosensor detecting pathological bile salt levels in serum from patients having undergone liver transplant, providing an output detectable by the naked-eye. The EMeRALD technology enables functional exploration of natural sensing modules and rapid engineering of synthetic receptors for diagnostics, environmental monitoring, and control of therapeutic microbes.

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“…While transfer to other proteobacteria like Salmonella may be direct, changes are needed for other desired bacteria with different promoter architectures. A promising host is Bacteroides thetaiotaomicron , gut bacteria in which others have built dCas9–sgRNA logic gates ( 66 )—their study included seven orthogonal sgRNA-promoter pairs, much like the pairs we used for our E. coli circuits. With functional prototypes in bacteria, it may be worth exploring construction in eukaryotic organisms like yeast where CRISPR-Cas has been much explored, though a complete circuit reconstruction will be required.…”

Section: Discussionmentioning

confidence: 99%

Toward a translationally independent RNA-based synthetic oscillator using deactivated CRISPR-Cas

Kuo

Yuan

Sánchez

et al. 2020

Nucleic Acids Research

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Abstract In synthetic circuits, CRISPR-Cas systems have been used effectively for endpoint changes from an initial state to a final state, such as in logic gates. Here, we use deactivated Cas9 (dCas9) and deactivated Cas12a (dCas12a) to construct dynamic RNA ring oscillators that cycle continuously between states over time in bacterial cells. While our dCas9 circuits using 103-nt guide RNAs showed irregular fluctuations with a wide distribution of peak-to-peak period lengths averaging approximately nine generations, a dCas12a oscillator design with 40-nt CRISPR RNAs performed much better, having a strongly repressed off-state, distinct autocorrelation function peaks, and an average peak-to-peak period length of ∼7.5 generations. Along with free-running oscillator circuits, we measure repression response times in open-loop systems with inducible RNA steps to compare with oscillator period times. We track thousands of cells for 24+ h at the single-cell level using a microfluidic device. In creating a circuit with nearly translationally independent behavior, as the RNAs control each others’ transcription, we present the possibility for a synthetic oscillator generalizable across many organisms and readily linkable for transcriptional control.

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Genetic circuit design automation for the gut resident species Bacteroides thetaiotaomicron

Cited by 96 publications

References 64 publications

Primary human colonic mucosal barrier crosstalk with super oxygen-sensitiveFaecalibacterium prausnitziiin continuous culture

Primary human colonic mucosal barrier crosstalk with super oxygen-sensitiveFaecalibacterium prausnitziiin continuous culture

A synthetic receptor platform enables rapid and portable monitoring of liver dysfunction via engineered bacteria

Toward a translationally independent RNA-based synthetic oscillator using deactivated CRISPR-Cas

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